Mutations in the STAT1‑interacting domain of the hepatitis C virus core protein modulate the response to antiviral therapy

Affiliations: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad 44000, Pakistan

Published online on:Tuesday, June 25, 2013

Pages:487-492DOI:10.3892/mmr.2013.1541

Abstract

RNA viruses, such as hepatitis C virus (HCV), have markedly error-prone replication, resulting in high rates of mutagenesis. In addition, the standard treatment includes ribavirin, a base analog that is likely to cause mutations in different regions of the HCV genome, resulting in deleterious effects on HCV itself. The N-terminal region of the core protein is reported to block interferon (IFN) signaling by interaction with the STAT1‑SH2 domain, resulting in HCV resistance to IFN therapy. In this study, mutations in the HCV core protein from IFN/ribavirin‑treated patients were analyzed, with particular focus on the N‑terminal domain of the HCV core which is reported to interact with STAT1. HCV PCR positive patients enrolled in this study were either undergoing pegylated IFN/ribavirin bitherapy and had completed 12 weeks of initial treatment or were treatment‑naïve patients. The HCV core protein was cloned and sequenced from these patients and mutations observed in the STAT1‑interacting domain of the core protein from treated patients were characterized using in silico interaction to depict the role of these mutations in disease outcomes. Our results suggest that the amino acids at positions 2, 3, 8, 16 and 23 of the HCV core protein are critical for core-STAT1 interaction and ribavirin-induced mutations at these positions interfere with the interaction, resulting in a better response of the treated patients. In conclusion, this study anticipates that HCV core residues 2, 3, 8, 16 and 23 directly interact with STAT1. We propose that IFN/ribavirin bitherapy‑induced mutations in the STAT1‑interacting domain of the HCV core protein may be responsible for the improved therapeutic response and viral clearance, thus amino acids 1-23 of the N-terminus of the core protein are an ideal antiviral target. However, this treatment may give rise to resistant variants that are able to escape the current therapy. We propose similar studies in responsive and non-responsive genotypes in order to gain a broader picture of this proposed mechanism of viral clearance.

Introduction

Hepatitis C virus (HCV) is a major public health
concern worldwide. Approximately 170 million people suffer from
chronic HCV and are at risk of developing cirrhosis and
hepatocellular carcinoma. In Pakistan alone, 10 million people are
infected with HCV and 50% of them are infected with the 3a subtype.
HCV is a small hepatotropic virus, and member of the
Flaviviridae family, infecting 170–180 million people
worldwide (1). Globally, 0.25–1.25
million new cases of HCV infection have been reported per year
(2). The core protein is the first
structural protein encoded by the HCV open reading frame (ORF),
consisting of 191 amino acids in its immature form. It is one of
the potential targets for specific drugs against HCV, as it is well
conserved in all HCV genotypes and interacts with a number of
cellular factors of the host immune system (3,4).
Expression of the HCV core protein results in suppression of type I
interferon (IFN) signaling leading to the reduction of
phosphorylated STAT1 (P-STAT1). HCV core protein and STAT1 are
reported to have a direct interaction involving residues in the
N-terminal portion of the HCV core (amino acids 1–23) (5). Mutations in the N-terminal of the
core protein are expected to modulate antiviral response in
general, as well as response to conventional pegylated interferon
(PEG-IFN) and ribavirin (PEG-IFN/ribavirin) combination therapy,
eventually leading to sustained virological response.

Emerging HCV resistance to the current standard
available treatment, PEG-IFN/ribavirin combination therapy, is of
great concern, as is its low response and toxicity (6). Although new direct acting antivirals
(DAAs) targeting HCV NS3–4A protease, namely telaprevir and
boceprevir, have shown an increase in the sustained virological
response (SVR) of up to 70% in patients infected with HCV genotype
1 (7), the conventional
PEG-IFN/ribavirin treatment remains part of the therapy. Clinical
studies have suggested that non-synonymous mutations are induced by
ribavirin monotherapy and thus increase IFN sensitivity (8). The SVR has been shown to be markedly
augmented by the addition of ribavirin to IFN monotherapy, with an
increase in the response rate and a reduction in the relapse rate
being observed (9). Mathematical
model applications have revealed viremic decay following
combination therapy (10).

In this study we propose a possible mechanism of
PEG-IFN/ribavirin-induced SVR. We suggest that PEG-IFN/ribavirin
therapy-induced amino acid changes in the N-terminus of the HCV
core are associated with viral clearance or persistence.

Materials and methods

Patient demographics

Patients with a positive PCR test for HCV, confirmed
by Atta-ur-Rahman School of Applied Biosciences (ASAB) Diagnostics,
were enrolled for this study under the approval of the Internal
Review Board (IRB) of ASAB, National University of Sciences and
Technology, Pakistan and a patient consent form was duly signed for
each patient. All patients were infected with genotype 3a, the most
prevalent genotype in Pakistan. The genotype of the patients was
determined using the method described by Ohno et al(11). The HCV patients selected for this
study were from two different groups. The patients in group A were
receiving treatment with pegylated interferon α-2a (PEG-IFN α2a)
180 μg/week and ribavirin 800 mg/day for 24 weeks. In group B,
patients that were recently diagnosed and had viral titer and
alanine transaminase (ALT) levels relatively close to those of
group A were selected (Table I).
The follow-up for the treated patients was carried out for 24 weeks
following the completion of treatment. The viral loads and ALT
levels of the patients were measured at 12-week intervals (Table I). Viral RNA was quantified using a
Bio-Rad RoboGene HCV amplification kit (Bio-Rad, Hercules, CA,
USA), whereas Microlab 300 (Merck, Germany) was employed for ALT
measurements. Sequencing of the isolated virus core gene was
performed after 12 weeks of PEG-IFN/ribavirin bitherapy. To compare
the mutations induced by ribavirin, the HCV core gene was also
cloned and sequenced from HCV-infected patients without liver
complications and who had not received any treatment.

[i] The HCV core gene
was amplified from the patient sera. In the treated patients, the
core gene was amplified after the completion of 12 weeks of therapy
and viral titer and alanine transaminase (ALT) levels were recorded
at an interval of 12 weeks.

PCR amplification cloning and sequencing
of HCV core gene

For PCR amplification of the core gene, viral RNA
was extracted from patient serum by using an RNA extraction kit
(Qiagen, Hamburg, Germany) according to the manufacturer's
instructions. The primers used for cDNA synthesis and PCR
amplification were: 5′-AAA GAA TTC GCC ACC ATG CTA GAG TGG CGG AAT
ACG TCT GGC C-3′ (sense) and 5′-CCC GCG GCC GCT TAA CTG GCT GCT GGA
TGA ATT AAG C-3′ (antisense). Purified PCR products were cloned in
PCRII TOPO Cloning vector (Invitrogen, Singapore) as instructed by
the manufacturer. Two clones from each patient were subjected to
sequencing using a CEQ 8000 genetic analysis system (Beckman
Coulter, Miami, FL, USA) as described previously (12). Sequences from the present patients
were aligned with reference isolates (Fig. 1). The aligned sequences of HCV core
(>3000 sequences) from the European database (http://euhcvdb.ibcp.fr/euHCVdb/) were analyzed to
check the conservation of the residues involved in core-STAT1
interactions in the N-terminus of the core gene.

Molecular modeling of HCV core gene and
its in silico interaction with STAT1

The HCV core gene consensus sequence of all 16
clones from the untreated patients was submitted to the I-TASSER
online web server (13) for
molecular modeling and the model with the highest C-value was
selected for further analysis. The model was refined with energy
minimization by subjecting it to ionized water box and
physiological concentrations. The AMBER 99 force field was used to
minimize its energy after protonation of the system by fixing its
charges and lone pairs. The minimized model was extracted from the
solvent system and was docked with the STAT1 protein (pdb id 1yvl).
The protein interaction between core and STAT1 was studied using
the HADDOCK web server (14).
Based on previous studies, residues 1–23 of the core were selected
as the active site and residues 577–684 of STAT1 were selected as
passive residues for this interaction. Different contact types,
including ionic cutoff 4.5, hydrophobic cutoff 4.5, hydrogen bonds
and disulfide cutoff 2.5, were evaluated between the core and STAT1
using the default bond angles used in the Molecular Operating
Environment (MOE). The sequence separation used was 4 residues
apart. Histidine was selected as it is basic in character whereas
methionine was characterized as hydrophobic in nature. The
mutations observed at core residues 2, 3, 8, 16 and 23 (Fig. 1) were incorporated into the in
silico interaction model. In order to investigate the further
significance of the observed mutations in the core
STAT1-interacting domain (amino acids 2, 3, 8, 16, 23) two way
ANOVA was applied on mutational data from treated and untreated
core residues. A P-value <0.05 was considered to indicate a
statistically significant difference.

Results

Sequence analysis

For sequence comparison, H77 genotype 1a was taken
as a reference strain for amino acid positioning. NZL1 and K3a
isolates of GT3a were taken as references for sequence analysis
(Fig. 1). Notably, as compared
with reference isolates and clones from untreated patients, few
major differences were observed in the N-terminal region of the
core. Residues 2, 3, 8, 16 and 23 were frequently mutated in
treated patients as compared with untreated patients (Fig. 1; Table II) and significant differences
(P<0.001) were recorded at positions 2, 3, 8 and 23.
Comprehensive analysis of the aligned core sequences reported in
the European database showed that residues 2, 3, 8 and 23 are well
conserved across all genotypes (Table III). Position 16 was not well
conserved and the mutations observed had no effect on core-STAT1
interactions.

Table II

In vivo mutations observed in
the hepatitis C virus core proteins from treated patients and their
effects on STAT1 interaction.

Table II

In vivo mutations observed in
the hepatitis C virus core proteins from treated patients and their
effects on STAT1 interaction.

Core residues

Mutations

Contact
alteration

2

S/R

No contacts
observed

3

T/L,I

No contacts
observed

7

P/L

Contact established
with Val 642 and Ile 647 of STAT1

8

R/Q

No contacts
observed

16

S/N,I

Contact conserved
either N/I with Asp 627

23

K/R

No contact
observed

[i] Contact report is
based on the results achieved from the in silico interaction
of the core with STAT1 using the HADDOCK web server.

[i] %variability was
calculated by comparison of 3,498 core sequences from the European
HCV database. Number indicated with mutated residue is the number
of times the mutation reported in 3498 sequences.

In silico characterization of the STAT1
binding domain of the core protein

Sequence variations observed in the present study
were mostly found in the N-terminus of the protein, a region which
has been previously shown to interfere with IFN signaling by
interacting with the STAT1-SH2 domain (15). We therefore investigated how these
amino acid changes potentially affect core-STAT1 interactions. For
this purpose, an in silico approach based on prediction of
molecular docking was used. Although the structure of STAT1 is
known, the HCV core protein structure has not been reported. We
therefore started by determining a structural model for the HCV
core protein (Fig. 2A). The
characterization of interaction contacts between the HCV core and
STAT1 were then determined (Fig.
2B; Table II) and the contact
details of the interacting residues are provided in Table III. Based on our molecular
modeling approaches, amino acids S, T, Q and K at positions 2, 3, 8
and 23, respectively, appear critical for core-STAT1 interaction.
Changes in these residues, as observed in some of our clones,
resulted in loss of contact between core and STAT1 (Fig. 2; Tables II and III). The follow-up data and the
core-STAT1 docking results clearly correlate the SVR observed in
six out of eight patients that carried observed mutations.
Follow-up of the untreated patients was not conducted, as they were
recommended for treatment.

Discussion

The mechanism of viral persistence and clearance has
not been well elucidated. Viral capsid proteins have been proposed
previously as targets for anti-viral drugs, as they are well
conserved across the 6 major genotypes (16). In the current study, the HCV core
gene was amplified from the serum of patients that were undergoing
PEG-IFN/ribavirin treatment for 12 weeks and from treatment-naïve
patients. As the core quasispecies tends to be conserved during
acute HCV infection (17), in the
present study patients without liver complications and at a
relatively early stage of disease were enrolled and thus a more
conserved core gene was anticipated.

Notably, as compared with isolates from untreated
patients (Fig. 1), few significant
differences were observed in the N-terminal region of the cores
from treated patients. Comprehensive analysis of the aligned core
sequences reported in the European database showed that residues 2,
3, 8 and 23 are well conserved across all genotypes. Amino acid
changes in this part of the protein are known to modulate viral
assembly or core interactions with host factors (18). The N-terminal region of the core
(amino acids 1–23) has been shown to block IFN signaling by
interaction with the STAT1-SH2 domain that plays a significant role
in HCV resistance to IFN therapy (19). In silico molecular docking
was used to observe the potential effects of these changes on the
core-STAT1 interaction. For this purpose, the HCV core protein
structure was modeled (Fig. 2A)
and the interaction contacts between the HCV core and STAT1 were
determined (Fig. 2B). The contact
details of the interacting residues are provided in Table II. Based on our molecular modeling
approaches, amino acids S, T, Q and K at residues 2, 3, 8 and 23
appear critical for core-STAT1 interaction. Changes in these
residues, as observed in the majority of our clones from treated
patients, resulted in a loss of contact between the core and STAT1.
Mutations at similar positions were rarely reported in the HCV
database and these residues tend to be conserved among various
genotypes.

Follow-up information (Table I) revealed that the core mutations
observed in six patients at critical residues resulted in a loss of
contact with STAT1, thus ensuring better antiviral response and
facilitating viral clearance. However, two of the patients,
patients 1 (PT1) and 2 (PT2), showed no mutation at these
positions. These two patients were non-responders and discontinued
therapy after six months (Table
I). Notably, in patient 4 (PT4), the virus had counteracted the
loss of the STAT1 interaction at position 8 by a P>L shift at
core position 7 that resulted in the establishment of a new
interaction with Val 642/Ile 647 of STAT1 (Table III). This new contact may
modulate STAT1 signaling and a relapse may occur following the
accumulation of the resistant variant. An early virological
response reported for genotype 3a was not evident in the current
study, possibly due to the small sample size; however, the
identification of non-responders is not unusual for genotype 3a. We
have recently recorded a significant difference in the mutation
rate of HCV glycoprotein E2 in treated vs. untreated patients and
have observed for the first time a glycosylation position shift in
envelope protein E2 that results in antibody escape variants,
giving the virus a chance to survive following the therapeutic
response (unpublished data).

Previous reports have indicated that amino acid
substitutions at position 70 and/or 91 in the HCV core protein
region of patients infected with HCV-1b are pretreatment predictors
of a poor virological response to PEG-IFN/ribavirin combination
therapy and telaprevir/PEG-IFN/ribavirin triple therapy (20,21).
In all patients included in this study, the core position 70 was
occupied by arginine as reported for genotype 1b and should favor
the treatment response, however the failure of patients 1 and 2 to
respond to treatment suggest that there may be more than one
predictor of therapeutic outcomes. Core residue 91 appears to be
genotype-specific and thus may contribute to the genotype-specific
antiviral response to IFN/ribavirin therapy. Another study,
however, described that the ribavirin monotherapy-induced mutagenic
effect, studied in the context of the NS3 and NS5B regions of HCV,
was reduced in patients receiving PEG-IFN and ribavirin combination
therapy, possibly due to the antiviral action of IFN (22). In the current study, since the
ribavirin monotherapy was not included due to its absence from the
general medical practices prevailing in Pakistan, there is a
possibility that certain other error mutations may have been
immediately eliminated by the concurrently administered IFN. This
may account for the relatively small number of mutations observed
in the current study, despite the presence of a mutagenic analog in
the combination bitherapy.

In conclusion, this study suggests that
IFN/ribavirin bitherapy-induced mutations in the STAT1-interacting
domain of the HCV core protein may be responsible for the improved
therapeutic response and viral clearance, at least in the GT3a
genotype, the most prevalent genotype in Pakistan. However, this
treatment may give rise to resistant variants that are able to
escape the current therapy. In addition, this study indicates for
the first time that residues 2, 3, 8, and 23 of the HCV core are
critical for the core-STAT1 interaction and we propose these
residues as a potential target for antiviral drug design.

Acknowledgements

The authors acknowledge Dr Jean Dubuisson for his
help in writing and improving this manuscript and ASAB Diagnostics
for HCV patient enrollment and for the follow-up records. This
study constitutes partial fulfillment for the degree of Doctor of
Philosophy for Anjum S. from ASAB (former NCVI), National
University of Science and Technology, Islamabad, Pakistan. The
authors also acknowledge HEC Pakistan and French split PhD
fellowship (EGIDE) for supporting this study.